The long-term goal of this proposal is to understand the molecular mechanisms of brain injury in order to devise novel therapeutic approaches to traumatic brain injury (TBI) treatment. TBI is a result of both immediate mechanical disruption of brain tissue (the primary injury) and delayed (secondary) injury mechanisms. A hallmark of secondary brain tissue injury in TBI is mitochondrial dysfunction and release of mitochondrial pro-apoptotic proteins leading to neural cell death. Although the nature of the molecule that causes loss of mitochondrial integrity and function remains obscure, the membrane sphingolipid ceramide, a key regulator of cell-stress responses could play a role. Strong evidence implicates the production of endogenous ceramide in response to apoptotic stimuli as a universal element of apoptosis. Our preliminary data indicate excessive ceramide accumulation in brain after experimental TBI due to activation of ceramide biosynthesis. Importantly, we have identified several isoforms of ceramide synthase localized in highly purified cerebral mitochondria. We have also shown the ability of natural ceramide to inflict mitochondrial injury similar to that occurring in mitochondria after TBI. We hypothesize that TBI triggers activation of ceramide biosynthesis that results in excessive accumulation of ceramide in mitochondria leading to release of mitochondrial pro-apoptotic proteins and neural cell death. We plan to test our hypothesis by pursuing 2 specific aims: 1) Determine if activation of ceramide biosynthesis is required for the secondary brain damage after TBI and identify the mechanisms involved; 2) Determine the mechanisms underlying the ceramide-mediated mitochondrial injury in TBI. We will use a computer-controlled cortical impact mouse model of TBI. In the proposed studies, we will use an interdisciplinary approach combining protein expression and activity assays with magnetic resonance imaging (MRI), behavioral and treatment studies in transgenic and knockout mice. We will use a powerful new methodology, tandem mass spectroscopy, for accurate detection of natural ceramide species and ceramide synthase activity. The proposed studies will advance our understanding of the mechanisms of TBI and reveal specific targets that can provide the basis for developing new approaches for therapeutic intervention. Furthermore, the proposed studies will link a novel cellular mechanism of post-traumatic cell dysfunction and death to the clinically relevant assessment of the injury by MRI in order to develop the most meaningful outcome measures for successful rehabilitation of the head-injured patient.